8 chamber slides

hDPCs were plated in 8-chamber slides (SPL, Korea) at a density of 2 × 10³ cells per well and cultured in serum-free medium in the presence of DHT or vehicle control (methanol) for 5 h. Immunofluorescence staining of AR was performed as previously described [36 (link)]. Briefly, proteins were immunolabeled by incubating with anti-AR antibody (1:100, Cell Signaling Technology), anti-CRTH2/DP2 antibody (1:100, Novus) and anti-rabbit Alex Fluor 488 conjugated antibody (1:200; Invitrogen, OR, USA). Slides were examined under Axiovert 200 microscope (ZEISS, Germany).

Cells were stained in accordance with a standard cell staining protocol. Briefly, TE5, TE15, and KYSE70 cells were cultured on SPL cell culture slides, which are 8-chamber slides (SPL Life Science, Pocheon, Korea) for 24 h. Cells were subsequently fixed with 4% paraformaldehyde at room temperature for 20 min, permeabilized in 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 1 min, and incubated in blocking buffer containing 1% bovine serum albumin for 30 min. Cells were then incubated with the anti-AQP1 antibody at room temperature overnight. After three washes in PBS, cells were incubated with Alexa Fluor 488-labeled goat anti-mouse secondary antibodies at room temperature for 1 h. After three washes in PBS, cells were incubated with rhodamine phalloidin and 40,6-diamidino-2-phenylindole (DAPI) for 30 min. Slides were then mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA). The distribution of AQP1 proteins was examined using a BZ-X700 (Keyence, Tokyo, Japan).

DNA fragmentation in intracellular amastigotes was analysed by fluorescence microscopy using the In Situ Apotosis Detection Kit (Trevizen, USA). Mouse peritoneal macrophages (1 x 10⁶) were plated in 8-chamber slides (SPL, Korea) and incubated at 37°C in 5% CO2 overnight. Next day unattached cells were washed off and then infected with parasites at 1:20 ratio. After 3 h of incubation the unbound parasites were removed by washing thrice with PBS. The infected cells were then incubated for further 24 h post infection and then treated with KalsomeTM10, Ambisome and amphotericin B for 1h after which the cells were washed with PBS and then incubated in fresh medium for 72 h post infection. The coverslips were then washed twice with PBS and fixed with 3.7% formaldehyde. The slides were then stained using the In Situ Apoptosis Detection Kit and examined using fluorescence microscopy.